Nowadays, in many clinical and laboratorial diagnostics of biological samples, simple low cost systems are required, with the possibility to quantify very small amounts in a fast and reliable way.
Among these, strong interest is put on the identification of nucleic acids, DNA/RNA, and/or proteins, for instance antibodies and/or antigens related with certain disease.
Nucleic acids are the genetic material of any living organism, containing specific information that allows its complete characterization. Therefore, it is possible to identify characteristic sequences for each living being, from which relevant information can be obtained: identification of sequences; identification of mutations which can cause diseases; detection of pathogenic agents such as bacteria and virus, etc [1].
Most of the known characterization techniques for DNA/RNA sequences are based on the selective and specific hybridization of a small oligonucleotide (probe) with the complementary DNA sequence (target). Nowadays, fluorescence or radioactive methods are the most used for the detection of specific sequences by hybridization. However, it is verified that these techniques are expensive and extremely slow [2, 3]. In addition, hybridization techniques need a significant amount of target to obtain a signal. Following this procedure these techniques are mainly susceptible to be applied after an amplification process of the nucleic acid from the sample in question, through the technique of polymerase chain reaction—PCR. PCR allows an amplification of the number of DNA molecules available, mimmetizing what happens in the multiplication process of cells in an organism.
New real time amplification techniques (for example Real-time PCR) offer a high automation level and decrease the time necessary for amplification and detection. With the use of these techniques, it is also possible to obtain quantitative results.
Nevertheless, in spite of the high costs associated with the equipment and for testing, these technologies present a great disadvantage, which prevents a broader use in laboratories—the sample handling, since highly purified samples are needed, which consequently requires highly specialized personal and equipped laboratories [4] [5].
More recently, DNA chips (micro-groups of integrated sensors) have earned some popularity. Their largest application has been in gene expression studies, where the chips offer simultaneous analyses of several genes for a single sample. Basically, this technique is based on simultaneous hybridization of a high number of samples with minimum amounts of sample.
In spite of all this, the amplification step is still required. Additionally, the chip content is still a problem to be solved, besides this to be a costly technology, since these chips are not reusable [6].
Several calorimetric methods for nucleic acids detection have been developed [7-10]. Some of these methods are based on the optical properties (plasmon surface resonance) of gold or other metal nanoparticles [11-12], function of their form and size.
These nanoparticles of gold or other metal, such as silver or silver-gold alloys, are extremely sensitive to the changes of the medium, presenting a colour variation, from red to blue in the case of gold. The colour variation can be the result of the aggregation of several particles, for instance, by the action of a non complementary DNA chain.
The colour change is a macroscopic response originated from a nanometric scale phenomenon, where DNA/RNA can behave in a complementary or non complementary manner. For each one of these reactions a different sensor system response exists for the absorption of incident light.
By doing so, the hybridization of DNA or RNA probes linked to particles of gold for the identification of specific sequences is a low cost and easy to use technique, which could be an alternative to conventional methods. Even though, the technique based on gold nanoparticles is quite simple and low cost one, it still requires the need to register the colour variation. Even more, if the target presents just a small colour change in relation to the probe, the deviation of the maximum (colour) peak can only be detected through equipment with a high sensitivity, involving also some limitations (such as the large amount of biological liquid required and the need of a high spectrum resolution).
On the other hand, besides the detection of the deviation in relation to the peak, these techniques are unable to supply quantified information, in terms of the intensity variation associated with one given colour or colour deviation.
Presently, there are several documents that describe related techniques in this area: so, the document corresponding to the patent US2006127931 refers to a detection process of the transmitted light associated to several nano-cavities. In this case the confinement of the light, associated to the areas where the nanoparticles are located, it is detected by measuring the reduction of the light transmitted through a photonic structure. This process is optimized for the range of wavelengths between 1450 and 1600 nanometers. The process of nano-cavities and wave guide is not considered in the present invention.
Patent document DE102004015272 is related to bio-sensors that use CMOS (complementary metal-oxide-semiconductor) technology which is intended to determine the presence of DNA that hybridizes with target molecules placed on the photodiode. By this way it is not possible to re-use the sensor, nor it is possible to quantify the signal, nor is it possible to determine the colour deviation. Even more, it does not use nanoprobes of metallic particles in the detection process. The probes/samples analysed are linked to an electrode which generates an electrical signal. The structure is different from the one proposed in this invention, since it is fully based on CMOS technology.
Patent document US2005046847 deals with a method for optical illumination and DNA detection considered of low cost and of fast response. The system is based on the sweeping of integrated groups of sensors based on micro-electromechanical systems. In this invention, the light sources to be used are multiple and commuted amongst themselves, so that the same bio-sensor can be illuminated by more than one light source, or a light source can illuminate several bio-sensors. Besides, it uses filters for discrimination of the different wavelengths and/or discrimination of the polarization of the light beam. Finally, it can still include micro-lenses to focus the incident light. However this technology does not apply to the nanoparticles component and it differs from the present invention on the following aspects: the optical properties of the sample in study have to be analyzed after optical illumination, therefore a post-processing/analyses of the sample is necessary. It is not present in the method the application of metal/gold nanoparticles. The structure is formed by two matrices, one containing the light sources and the other containing the detectors, therefore it is different to the present invention.
The document WO0075276 refers to a device for DNA detection based on a gallium arsenide sensor. In this case, the DNA probe is directly absorbed on the surface of an upper layer of the sensor, which it is a degenerated semiconductor conductive layer, or an insulating or semi-insulating layer. The detection is made by hybridization of the DNA with the sensor and controlled by the variation of the current after application of an electric field, or vice-versa. The material and structure used are also different. Therefore, this system is completely different from the proposed one because it is based on the change of conductivity of the medium.
The document DE10142691 claims a device for biochemical reaction detection and measurement by light transmission through pores of the reaction substrate. This invention uses porous silicon and detection by illumination and, for that reason it differs substantially from the invention claimed in the present document.
The document WO2004044549 presents methods and compositions for detection, in which a universal detector, containing probes, is incubated with marked molecules. This method makes use of markers but does not specify the detection method used (calorimetric, impedance, etc.).
The document EP0667398 describes methods and devices for detection of specific sequences of DNA without having to use chemical markers, using only hybridization techniques. The present invention differs from the published document since an automatic method for the detection of the hybridization does not exist, therefore no illumination source exists, nor a device for capturing the reactions that happen. The hybridization has to be registered visually.
The document DE10161529 describes a bio-sensor for the registration and identification of DNA molecules, with the immobilization of the sample performed by a cavity covered with gold that works as a probe, which contains a photodiode. By illumination of the sample, the optical signals are detected by a photodiode. This invention differs substantially from the proposed present invention especially in respect to the probes connected to the detector and also on the design of the structure. Also the light source cannot illuminate directly the detection unit, which it is different from the present invention.
The document EP0248690 deals with methods and devices for the identification of viral nucleic acids in biological environments. Although it refers to the possibility of using a colorimetric method, it is very different from the present invention by its structure and detection method.
The document CN1661094 relates to a method for gene mutation detection by the combination of: amplification of specific alleles, gold nanoprobes and colorimetric methods. However, the described process differs substantially of the one presented here since it does not specify the detection method of the hybridization. The hybridization will be registered visually. No illumination source exists, nor a device for capturing the reactions that happen.
The document US2006014237 presents a detection system for biological agents by combination of: light emitting diodes for intermittently exposing the samples to electromagnetic radiation, circuitry to provide the exposure and photodetectors for detecting the fluorescent emissions resulting for the exposure to the radiation. The described system differs to the one presented here since it not used for nucleic acids detection, so no hybridization process or metal probes are used and the photodetectors are used for fluorescence detection.
The document US2006028955 presents an optical analyzer for biological, chemical and biochemical samples to measure the level of fat and sugar in blood. The system uses laser diodes and photodetectors enabling the analyzer to perform absorbance measurements analyses. This system differs from the one presented here since it is not used for nucleic acid detection, therefore no use of hybridization techniques nor metal nanoprobes.
Analyses of biological samples, as for instance in the specific case of DNA/RNA and proteins, are used in forensic analysis, clinical diagnosis and laboratorial markets and research. Nowadays, these techniques are used for the diagnosis of infectious diseases, although cancer diagnosis and genetics (research) represent relevant areas of application. The main industries that currently use detection methods of biological samples, as for instance in the specific case of DNA/RNA and proteins are:                Defence Organizations->issues associated to bio-safety have provoked the increase in the search for fast and reliable solutions for DNA/RNA tests, especially in the United States.        Medical institutions->they are used for medical diagnosis, namely for screening of genetic diseases or identification of pathogenic agents.        R&D Organizations->Many molecular tests are done using DNA detection for research purposes.        Health centres and laboratories for screening and analysis of infectious diseases.        World health organization.        Governmental and non-governmental organizations for detection, combat and eradication of pathogenic diseases.        
Besides these industries, that already use detection methods of biological samples, as for instance, in the specific case of DNA/RNA and proteins, new segments will also be able to make use of these tests, such as:                Food industry->Quality control of products, through sampling of the products and laboratory analyses.        Pharmaceutical industry, for quantification tests of the drug-delivery action.        Veterinary/Agricultural industry->for the detection of pathogenic agents, namely in the case of the agricultural industry, for the detection of genetically modified organisms.        
Tests of biological samples, as for instance, in the specific case of DNA/RNA and proteins are already used in a wide range field of applications, however, due to the associated costs and handling difficulties of the samples, the field of applications is limited.
The present invention has the objective to develop a simple and cheap system that will allow the ‘substitution’ of any detection system (for instance, a conventional spectrophotometer) by a photodetector of high sensitivity, in a wide range of wavelengths from the infrared to the ultraviolet, capable of supplying a qualitative and quantitative information based on the specific and selective hybridization of probes functionalized with gold nanoparticles, or any other metal, for the detection of biological samples, as for instance, in the specific case of specific sequences of DNA/RNA, in a faster way and, at least, as reliable as the existing methods.
This new method, combining two technologies, can lead to significant reductions in costs and time per analyses of biological samples, as for instance, in the specific case of DNA/RNA and, therefore, allow this type of molecular tests to be accomplished in practically any part of the world.
Besides, it is also possible to have a self-powered version of the all sensor system, for fast test application of the type Yes/No, associating the photodetector to a component operating in the photovoltaic mode, presenting a portability characteristic to the system.